Ninhydrin - Collidine Reagent

Dissolve 0.3 g ninhydrin (2,2-dihydroxy-l,3-indanedione) in 95 ml 2-propanol and add 5 ml collidine (2,4,6-trimethylpyri-dine) and 5 ml acetic acid (96%). 

If the solvents are not sufficiently pure the solution may only be kept for a short time. 

The course of the reaction has not been fully explained one possible route is  

The chromatogram is freed from mobile phase, immersed for 1 s in the reagent solution and then heated to 95—120°C for 5 — 10 min. After 15 min stabilization time mainly reddish but sometimes blue substance zones appear on a pale background. 

Note The reagent can be employed on silica gel and cellulose layers and the dipping solution can also be employed as a spray solution. The addition of collidine 

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Fig. 1 Comparative recordings of the reflectance scans of a mixture of phenylethylamine (1), tyramine (2), serotonin (3) and histamine (4) A. ninhydrin reagent (q.v.), B. ninhydrin — collidine reagent.

The mechanism of the fluorescence reaction has not been elucidated. See Volume 1 a Ninhydrin — Collidine Reagent for the ninhydrin reaction. 

Figure 10.17 Two-dimensional high voltage electrophoresis and chromatography of amino acids. Paper high voltage electrophoresis (4000 V) in an acetic acid-formic acid buffer at pH 2.0 in the first dimension followed by descending chromatography in the second dimension in an n-butanol-acetic acid-water solvent (12 3 5). The spots were visualized with a ninhydrin-collidine reagent.

Ninhydrin — Collidine Reagent Ninhydrin — Collidine Reagent 355... 

The ninhydrin colour reaction has proved very useful in qualitative work and is widely used in the visualization of amino acid bands after electrophoretic or chromatographic separation of mixtures. The reagent used in such circumstances is usually prepared in ethanol and, if 2,4,6-collidine is added, the variations in colour produced by different amino acids will aid their identification (Table 10.6). 

Ninhydrin, 0.2% (w/v), is dissolved in ethanol and stored in a refrigerator. Just prior to use, 0.5 ml of 2,4,6-collidine is added to each 50 ml of the ninhydrin solution. Color development can be hastened by heating the dipped plate with a warm air gun. This reagent gives distinctive colors for a large number of amino acids (Table 2). 

Use ninhydrin reagent without collidine (Section 4.7.2) followed by Ehrlich s reagent (Section 4.7.5) to detect citrulline and hydroxyproline. Citrulline gives a bluish pink color hydroxyproline is pink. 

Polychromatic reagents Moffat and Lyttle (42) developed a polychromatic ninhydrin reagent. It consisted of (a) ninhydrin (0.2%) in ethanol (50 ml) + acetic acid (10 ml) + 2,4,6-collidine (2 ml) and (b) a solution of copper nitrate (1.0%) in absolute ethanol. The two solutions are mixed in a ratio of 50 3 before use. Krauss and Reinbothe replaced ethanol by methanol (43) and also achieved polychromatic amino acid detection by joint application of ninhydrin and primary, secondary, or tertiary amines. The layers were first sprayed with diethylamine, dried for 3 min at 110°C, cooled, and then sprayed with 0.2% methanolic ninhydrin and heated for 10 min at 110°C, when the spots of amino acids appeared on a pale blue background. Use of ninhydrin (0.27 g), isatin (0.13 g), and triethylamine (2 ml) in methanol (100 ml) gave spots of amino acids on a yellow background. 

The following materials may be obtained as reagent or other similar quality grade, and used without further purification chloroform, 2,4,6-collidine, dichloromethane, dicyclohexylcarbodiimide, l-(3-dimethyl-aminopropyl)-ethylcarbodiimide,4-dimethylaminopyridine, ethanol, 1 -methylimidazole, ninhydrin.p-nitrophenol, pentachlorophenol, piperidine, succinic anhydride, trichloroacetic acid, triethylamine, and toluene. The following reagents may be used as purchased, but must be stored tightly sealed to avoid undue exposure to moisture acetic anhydride, 3-aminopropyltriethoxysilane, and trimethylsilyl chloride. 

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